Assays directed to the detection and quantification of physiologically significant materials in biological fluid and tissue samples are important tools in scientific research and in the health care field.
Several different types of assay have been developed that are capable of detecting relatively high concentrations of components of common biological samples such as human serum (Zhang T. H., et al., “Detection for anti-hantavirus IgM in patient serum with silver enhanced dot immunogold filtration assay,” Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi 2000 September; 14(3):266-7). Such assays include high-resolution agarose gel electrophoresis and test procedures based on the catalytic activity of endogeneous enzymes (Bhattacharyya S. P., et al., “Structural analysis of DNA cleaved in vivo by bacteriophage T4 terminase,” Gene 1994 Aug. 19; 146(1):67-72; Gaillot O.,et al, “Molecular characterization and expression analysis of the superoxide dismutase gene from Streptococcus agalactiae,” Gene 1997 Dec. 19; 204(1-2):213-8; Trigueros S., et al., “Novel display of knotted DNA molecules by two-dimensional gel electrophoresis,” Nucleic Acids Res 2001 Jul. 1; 29(13):E67-7). These methods generally do not have the sensitivity required to detect and quantify the numerous other physiologically important sample constituents which may be present at very low concentrations (e.g., endogeneous molecules intimately involved in cellular regulation (hormones, steroids, biochemical messengers); basic structural components of the organism (amino acids, proteins, polysaccharides); genetic material (DNA, RNA); vitamins, drugs and drug metabolites; toxins, pathogens and substances generated by the immune system).
Bioconjugates, such as protein-oligonucleotide conjugates, are employed in a wide variety of molecular biology applications (see, Reddy, U.S. Pat. No. 5,648,213). They are used in diagnostic assays to improve assay sensitivity (U.S. Pat. No. 6,197,513 (Coull, et al.). Such conjugates have traditionally been prepared by methods, such as glutaraldehyde crosslinking, maleimide-thiol coupling (Ghosh S S, et al., “Use of maleimide-thiol coupling chemistry for efficient syntheses of oligonucleotide-enzyme conjugate hybridization probes,” Bioconjug Chem 1990 January-February; 1(1):71-6), isothiocyanate-amine coupling (Brandtzaeg, 1973, Scand. J. Immunol. 2: 273-290; Loken and Herzenberg, 1975, Annals N.Y. Acad. Sci. 254: 163-171; U.S. Pat. No. 5,648,213 (Reddy, M. P.); Keller, G. H., et al., “DNA Probes,” MacMillan Publishers Ltd., 1989), and Schiff base formation/reduction.
Bioconjugates have served a variety of purposes in modem research. For example, bioconjugates such as oligonucleotides conjugated to antibodies or enzymes have been used as hybridization probes in immunoassays (U.S. Pat. No. 5,648,213 (Reddy, M. P.); Ghosh S S, et al., “Use of maleimide-thiol coupling chemistry for efficient syntheses of oligonucleotide-enzyme conjugate hybridization probes,” Bioconjug Chem 1990 January-February; 1(1):71-6; Keller and Manak, DNA Probes, 2nd Edition (Stockton Press, New York, 1993; Milligan et al, J. Med. Chem., 36: 1923-1937 (1993); Drmanac et al, Science, 260: 1649-1652 (1993); Bains, J. DNA Sequencing and Mapping, 4: 143-150 (1993)). Oligonucleotide-antibody conjugates have also been used as probes in the development of sensitive nucleic acid-based diagnostic assays (Martin R., et al., “A highly sensitive, nonradioactive DNA labeling and detection system,” 13: Biotechniques 1990 December; 9(6):762-8) (Podbielski A, et al., “Identification of group A type 1 streptococcal M protein gene by a non-radioactive oligonucleotide detection method,” 14: Med Microbiol Immunol (Berl) 1990; 179(5):255-62; Carpenter W. R., et al., “A transcriptionally amplified DNA probe assay with ligatable probes and immunochemical detection,” 9: Clin Chem 1993 September; 39(9):1934-8). Other bioconjugates, such as isothiocyanates (ITCs) conjugates, are used in bioassays as versatile chemopreventive agents (Chung E. L., “Chemoprevention of lung cancer by isothiocyanates and their conjugates in A/J mouse,” Exp Lung Res 2001 April-May; 27(3):319-30). Protein-polysaccharide conjugates with reciprocally enhanced immunogenicity have been used in the development of combination vaccines (Gupta R. K., et al, “Adjuvants for human vaccines—current status, problems and future prospects,” Vaccine 1995 October; 13(14):1263-76).
Despite the promise that bioconjugates hold for improving assay sensitivity and simplifying nucleic acid detection schemes, they have not become common place tools in molecular biology and diagnostic applications. The preparation of bioconjugates involves multiple steps that require the protein, oligonucleotide, or both, to be modified with the appropriate linking moiety and then purified before being combined and reacted with each other. Often the modification reaction results in an unstable reactive enzyme or oligomer intermediate that must be purified and used immediately. For these and other reasons the yield of conjugate is highly variable when these techniques are used. Furthermore, reaction times are lengthy, and several purification steps are generally needed to obtain a purified conjugate. Finally, in most instances a portion of the enzymatic activity is lost due to the nature of the chemical reactions, lengthy reaction times, and numerous purification steps.
For these reasons direct conjugates are expensive and difficult to make with reproducible results. This has prevented them from becoming commonplace tools in molecular biology and diagnostic applications despite the promise they hold for improving assay sensitivity and simplifying nucleic acid detection schemes.
Despite such efforts, a need continues for methods that provide a more efficient conjugation of an oligonucleotide to an antibody to improve the sensitivity of oligonucleotide-antibody conjugates as reagents in immunoassays. The present invention is directed to such a need.